Cyclic hexapeptides having antibiotic activity

ABSTRACT

This invention relates to new polypeptide compounds represented by the following formula (I):                    
     wherein 
     R 1  is as defined in the description or a salt thereof which have antimicrobial activities (especially, antifungal activities), inhibitory activity on β-1,3-glucan synthase, to process for preparation thereof, to a pharmaceutical composition comprising the same, and to a method for the prophylactic and/or therapeutic treatment of infectious disease including  Pneumocystis carinii  infection (e.g.,  Pneumocystis carinii  pneumonia) in a human being or an animal.

TECHNICAL FIELD

The present invention relates to new polypeptide compound and apharmaceutically acceptable salt thereof which are useful as amedicament.

BACKGROUND ART

In U.S. Pat. No. 5,376,634, U.S. Pat. No. 5,502,033, etc., there aredisclosed the polypeptide compound and a pharmaceutically acceptablesalt thereof, which have antimicrobial activities (especially antifungalactivity).

DISCLOSURE OF INVENTION

The present invention relates to new polypeptide compound and apharmaceutically acceptable salt thereof.

More particularly, it relates to new polypeptide compound and apharmaceutically acceptable salt thereof, which have antimicrobialactivities [especially, antifungal activities, in which the fungi mayinclude Asperaillus, Cryptococcus, Candida, Mucor, Actinomyces,Histoplasma, Dermatophyte, Malassezia, Fusarium and the like.],inhibitory activity on β-1,3-glucan synthase, and further which areexpected to be useful for the prophylactic and/or therapeutic treatmentof Pneumocystis carinii infection (e.g. Pneumocystis carinii pneumonia)in a human being or an animal, and further the toxicity of which areexpected to be much lower in comparison with the former polypeptidecompounds, to a process for preparation thereof, to a pharmaceuticalcomposition comprising the same, and to a method for the prophylacticand/or therapeutic treatment of infectious diseases includingPneumocystis carinii infection (e.g. Pneumocystis carinii pneumonia) ina human being or an animal.

The object polypeptide compound of the present invention is new and canbe represented by the following general formula [I]: (SEQ ID NO: 1)

wherein R¹ is hydrogen or acyl group, or a salt thereof.

The polypeptide compound [I] of the present invention can be prepared bythe processes as illustrated in the following schemes.

wherein R_(a) ¹ is acyl group.

Suitable salt of the object compound [I] is a pharmaceuticallyacceptable salt such as conventional non-toxic mono or di salts andinclude a metal salt such as an alkali metal salt [e.g. sodium salt,potassium salt, etc.] and an alkaline earth metal salt [e.g. calciumsalt, magnesium salt, etc.], an ammonium salt, an organic base salt[e.g. trimethylamine salt, triethylamine salt, pyridine salt, picolinesalt, dicyclohexylamine salt, N,N-dibenzylethylenediamine salt, etc.],an organic acid addition salt [e.g. formate, acetate, trifluoroacetate,maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate,etc.], an inorganic acid addition salt [e.g. hydrochloride,hydrobromide, hydroiodide, sulfate, phosphate, etc.], a salt with anamino acid [e.g. arginine salt, aspartic acid salt, glutamic acid salt,etc.], and the like.

In the above and subsequent description of this specification, suitableexamples of the various definitions are explained in detail as follows:

The term “lower” is intended to mean 1 to 6 carbon atom(s), unlessotherwise indicated.

The term “higher” is intended to mean 7 to 20 carbon atoms, unlessotherwise indicated.

Suitable “acyl group” may be aliphatic acyl, aromatic acyl, heterocyclicacyl, arylaliphatic acyl and heterocyclicaliphatic acyl derived fromcarboxylic acid, carbonic acid, carbamic acid, sulfonic acid, and thelike.

Suitable example of the “acyl group” thus explained may be:

lower alkanoyl [e.g. formyl, acetyl, propionyl, butyryl, isobutyryl,valeryl, hexanoyl, pivaloyl, etc.] which may have one or more(preferably 1 to 3) suitable substituent(s) such as halogen (e.g.fluoro, chloro, bromo, iodo); aryl (e.g. phenyl, naphthyl, anthryl,etc.) which may have one or more (preferably 1 to 3) suitablesubstituent(s) like hydroxy, higher alkoxy as explained below, aforesaidaryl, or the like; lower alkoxy as explained below; amino; protectedamino, preferably, acylamino such as lower alkoxycarbonylamino (e.g.methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino,butoxycarbonylamino, t-butoxycarbonylamino, pentyloxycarbonylamino,hexyloxycarbonylamino, etc.); or the like; di(lower)alkylamino (e.g.dimethylamino, N-methylethylamino, diethylamino, N-propylbutylamino,dipentylamino, dihexylamino, etc.); lower alkoxyimino (e.g.methoxyimino, ethoxyimino, propoxyimino, butoxyimino, t-butoxyimino,pentyloxyimino, hexyloxyimino, etc.);

ar(lower)alkoxyimino such as phenyl(lower)alkoxyimino (e.g.benzyloxyimino, phenethyloxyimino, benzhydryloxyimino, etc.) which mayhave one or more (preferably 1 to 3) suitable substituent(s) like higheralkoxy as explained below, or the like; heterocyclicthio, preferably,pyridylthio, which may have one or more (preferably 1 to 3) suitablesubstituent(s) like higher alkyl (e.g. heptyl, octyl, 2-ethylhexyl,nonyl, decyl, 3,7-dimethyloctyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, 3-methyl-10-ethyldodecyl, hexadecyl, heptadecyl, octadecyl,nonadecyl, icosyl, etc.), or the like; heterocyclic group (e.g. thienyl,imidazolyl, pyrazolyl, furyl, tetrazolyl, thiazolyl, thiadiazolyl, etc.)which may have one or more (preferably 1 to 3) suitable substituent(s)like amino, aforesaid protected amino, aforesaid higher alkyl, or thelike; or the like;

higher alkanoyl [e.g. heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, lauroyl, tridecanoyl, myristory, pentadecanoyl, palmitoyl,10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl,icosanoyl, etc.], in which the preferred one may be (C₇-C₁₇)alkanoyl,and the most preferred one may be palmitoyl;

lower alkenoyl [e.g. acryloyl, methacryloyl, crotonoyl, 3-pentenoyl,5-hexenoyl, etc.] which may have one or more (preferably 1 to 3)suitable substituent(s) such as aforesaid aryl which may have one ormore (preferably 1 to 3) suitable substituent(s) like higher alkoxy asexplained below, or the like, or the like;

higher alkenoyl [e.g. 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl,3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.];

lower alkoxycarbonyl [e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl,hexyloxycarbonyl, etc.];

higher alkoxycarbonyl [e.g. heptyloxycarbonyl, octyloxycarbonyl,2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl,3,7-dimethyloctyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl,tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl,3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl,heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl,icosyloxycarbonyl, etc.];

aryloxycarbonyl [e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.];

arylglyoxyloyl [e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc.];

ar(lower)alkoxycarbonyl which may have one or more suitablesubstituent(s) such as phenyl(lower)alkoxycarbonyl which may have nitroor lower alkoxy [e.g. benzyloxycarbonyl, phenethyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-methylbenzyloxycarbonyl, etc.];

lower alkylsulfonyl [e.g. methylsulfonyl, ethylsulfonyl, propylsulfonyl,isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.];

arylsulfonyl [e.g. phenylsulfonyl, naphthylsulfonyl, etc.] which mayhave one or more (preferably 1 to 3) suitable substituent(s) such aslower alkyl as explained below, higher alkoxy as explained below, or thelike;

ar(lower)alkylsulfonyl such as phenyl(lower)alkylsulfonyl [e.g.benzylsulfonyl, phenethylsulfonyl, benzhydrylsulfonyl, etc.], or thelike;

aroyl [e.g. benzoyl, naphthoyl, anthrylcarbonyl, etc.] which may haveone or more (preferably 1 to 5) suitable substituent(s) such asaforesaid halogen; lower alkyl (e.g. methyl, ethyl, propyl, butyl,t-butyl, pentyl, hexyl, etc.); aforesaid higher alkyl; lower alkoxy(e.g. methoxy, ethoxy, propoxy, butoxy, t-butoxy, pentyloxy, hexyloxy,etc.) which may have one or more (preferably 1 to 10) suitablesubstituent(s) like aforesaid lower alkoxy, aforesaid halogen, aforesaidaryl, or the like; higher alkoxy (e.g. heptyloxy, octyloxy,2-ethylhexyloxy, nonyloxy, decyloxy, 3,7-dimethyloctyloxy, undecyloxy,dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,3-methyl-10-ethyldodecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy,nonadecyloxy, icosyloxy, etc.) which may have one or more (preferably 1to 17) suitable substituent(s) like aforesaid halogen, in which thepreferred one may be aroyl having (C₇-C₁₇)alkoxy, and the more preferredone may be benzoyl having (C₇-C₁₇)alkoxy, and the most preferred one maybe benzoyl having octyloxy; higher alkenyloxy (e.g. 3-heptenyloxy,7-octenyloxy, 2,6-octadienyloxy, 5-nonenyloxy, 1-decenyloxy,3,7-dimethyl-6-octenyloxy, 3,7-dimethyl-2,6-octadienyloxy,8-undecenyloxy, 3,6,8-dodecatrienyloxy, 5-tridecenyloxy,7-tetradecenyloxy, 1,8-pentadecadienyloxy, 15-hexadecenyloxy,11-heptadecenyloxy, 7-octadecenyloxy, 10-nonadecenyloxy, 18-icosenyloxy,etc.); carboxy;

aforesaid aryl which may have one or more (preferably 1 to 3) suitablesubstituent(s) like aforesaid higher alkoxy; aryloxy (e.g. phenoxy,naphthyloxy, anthryloxy, etc.) which may have one or more (preferably 1to 3) suitable substituent(s) like aforesaid lower alkoxy, or aforesaidhigher alkoxy; aroyl substituted with heterocyclic group which has arylhaving lower alkoxy; aroyl substituted with heterocyclic group which hasaryl having higher alkoxy;

ar(lower)alkenoyl substituted with aryl having lower alkoxy;ar(lower)alkenoyl substituted with aryl having higher alkoxy; aroylsubstituted with aryl which has aryl having lower alkoxy; aroylsubstituted with aryl which has aryl having higher alkoxy; and the like.

Suitable example of “aroyl” may include benzoyl, toluoyl, naphthoyl,anthrylcarbonyl, and the like, in which the preferred one may bebenzoyl.

Suitable example of “aryl” and “ar” moiety may include phenyl which mayhave lower alkyl (e.g., phenyl, mesityl, tolyl, etc.), naphthyl,anthryl, and the like, in which the preferred one may be phenyl.

Suitable example of “lower alkoxy” may include straight or branched onesuch as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,tert-butoxy, pentyloxy, tert-pentyloxy, neo-pentyloxy, hexyloxy,isohexyloxy, and the like.

Suitable example of “higher alkoxy” may include straight or branched onesuch as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy,nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy,hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, icosyloxy, andthe like,

in which the preferred one may be (C₇-C₁₇)alkoxy.

Suitable example of “heterocyclic group” and “heterocyclic” moiety mayinclude

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example,pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl,pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl,1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), tetrazolyl (e.g.,1H-tetrazolyl, 2H-tetrazolyl, etc.), etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example,pyrrolidinyl, imidazolidinyl, piperidyl, piperazinyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 4 nitrogenatom(s), for example, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3nitrogen atom(s), for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g.,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.), etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3nitrogen atom(s), for example, morpholinyl, syndonyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 oxygenatom(s) and 1 to 3 nitrogen atom(s), for example, benzoxazolyl,benzoxadiazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3nitrogen atom(s), for example, thiazolyl, isothiazolyl, thiadiazolyl,(e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,1,2,5-thiadiazolyl, etc.), dihydrothiazinyl, etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3nitrogen atom(s), for example, thiazolidinyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing 1 to 2 sulfur atom(s), for example,thienyl, dihydrodithiinyl, dihydrodithionyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 sulfuratom(s) and 1 to 3 nitrogen atom(s), for example, benzothiazolyl,benzothiadiazolyl, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom, for example, furyl,etc.;

saturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom, for example,tetrahydrofuran, tetrahydropyran, etc.;

unsaturated 3 to 8-membered (more preferably 5 or 6-membered)heteromonocyclic group containing an oxygen atom and 1 to 2 sulfuratom(s), for example, dihydrooxathiinyl, etc.;

unsaturated condensed heterocyclic group containing 1 to 2 sulfuratom(s), for example, benzothienyl, benzodithiinyl, etc.;

unsaturated condensed heterocyclic group containing an oxygen atom and 1to 2 sulfur atom(s), for example, benzoxathiinyl, etc.; and the like, inwhich the preferred one may be saturated 3 to 8-memberedheteromonocyclic group containing 1 to 4 nitrogen atom(s) andunsaturated 3 to 8-membered heteromonocyclic group containing 1 to 2oxygen atom(s) and 1 to 3 nitrogen atom(s).

Suitable example of “aroyl substituted with heterocyclic group which hasaryl having lower alkoxy” may be benzoyl substituted with saturated6-membered heteromonocyclic group containing 1 to 4 nitrogen atom(s)which has phenyl having (C₄-C₆)alkoxy, benzoyl substituted withunsaturated 5-membered heteromonocyclic group containing 1 to 2 sulfuratom(s) and 1 to 3 nitrogen atom(s) which has phenyl having (C₄-C₆)alkoxy or benzoyl substituted with unsaturated 5-memberedheteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3nitrogen atom(s) which has phenyl having (C₄-C₆)alkoxy, in which thepreferred one may be benzoyl substituted with piperazinyl which hasphenyl having (C₄-C₆)alkoxy, benzoyl substituted with thiadiazolyl whichhas phenyl having (C₄-C₆)alkoxy or benzoyl substituted with isoxazolylwhich has phenyl having (C₄-C₆)alkoxy, and the most preferred one may bebenzoyl substituted with piperazinyl which has phenyl having hexyloxy,benzoyl substituted with thiadiazolyl which has phenyl having hexyloxyor benzoyl substituted with isoxazolyl which has phenyl havingpentyloxy.

Suitable example of “ar(lower)alkenoyl substituted with aryl havinglower alkoxy” may be phenyl(C₃-C₆)alkenoyl substituted with phenylhaving (C₄-C₆)alkoxy, in which the preferred one may be phenylacryloylsubstituted with phenyl having pentyloxy.

Suitable example of “aroyl substituted with aryl which has aryl havinglower alkoxy” may be benzoyl substituted with phenyl which has phenylhaving (C₄-C₆)alkoxy, in which the preferred one may be benzoylsubstituted with phenyl which has phenyl having pentyloxy.

The process for preparing the object compound [I] or a salt thereof ofthe present invention is explained in detail in the following.

Process 1

The object polypeptide compound [Ia] or a salt thereof can be preparedby the fermentation process.

The fermentation process is explained in detail in the following.

The compound [Ia] or a salt thereof of this invention can be produced byfermentation of the compound [Ia] or a salt thereof-producing strainbelonging to the genus Coleophoma such as Coleophoma sp. F-11899 in anutrient medium in the presence of leucine.

(i) Microorganism

Particulars of the microorganism used for producing the compound [Ia] ora salt thereof is explained in the following.

The strain F-11899 was originally isolated from a soil sample collectedat Iwaki-shi, Fukushima-ken, Japan. This organism grew ratherrestrictedly on various culture media, and formed dark grey to brownishgrey colonies. Anamorph (conidiomata) produced on a steam-sterilizedleaf segment affixed on a Miura's LCA plate¹) or a corn meal agar plateby inoculating the isolate, while neither teleomorph nor anamorph formedon the agar media. Its morphological, cultural and physiologicalcharacteristics are as follows.

Cultural characteristics on various agar media are summarized inTable 1. Cultures on potato dextrose agar grew rather rapidly, attaining3.5-4.0 cm in diameter after two weeks at 25° C. This colony surface wasplane, felty, somewhat wrinkly and brownish grey. The colony center waspale grey to brownish grey, and covered with aerial hyphae. The reversecolor was dark grey. Colonies on malt extract agar grew morerestrictedly, attaining 2.5-3.0 cm in diameter under the sameconditions. The surface was plane, thin to felty and olive brown. Thecolony center was yellowish grey, and covered with aerial hyphae. Thereverse was brownish grey.

The morphological characteristics were determined on basis of thecultures on a sterilized leaf affixed to a Miura's LCA plate.Conidiomata formed on the leaf segment alone. They were pycnidial,superficial, separate, discoid to ampulliform, flattened at the base,unilocular, thin-walled, black, 90-160(-200) μm in diameter and 40-70 μmhigh. Ostiole was often single, circular, central, papillate, 10-30 μmin diameter and 10-20 μm high. Conidiophores formed from the lower layerof inner pycnidial walls. They were hyaline, simple or sparinglybranched, septate and smooth. Conidiogenous cells were enteroblastic,phialidic, determinate, ampulliform to obpyriform, hyaline, smooth,5-8×4-6 μm, with a collarette. The collarettes were campanulate tocylindrical, and 14-18×3-5 μm. Conidia were hyaline, cylindrical,thin-walled, aseptate, smooth and 14-16(-18)×2-3 μm.

The vegetative hyphae were septate, brown, smooth and branched. Thehyphal cells were cylindrical and 2-7 μm thick. The chlamydospores wereabsent.

The strain F-11899 had a temperature range for growth of 0 to 31° C. andan optimum temperature of 23 to 27° C. on potato dextrose agar.

The above characteristics indicate that the strain F-11899 belongs tothe order Coelomycetes^(2), 3), 4)). Thus, we named the strain“Coelomycetes strain F-11899”.

TABLE 1 Cultural characteristics of the strain F-11899 Medium Culturalcharacteristics Malt extract agar G: Rather restrictedly, 2.5-3.0 cm(Blakeslee 1915) S: Circular, plane, thin to felty, olive brown (4F5),arising aerial hyphae at the center (yellowish grey (4B2) R: Brownishgrey (4F2) Potato dextrose agar G: Rather rapidly, 3.5-4.0 cm (Difco0013) S: Circular, plane, felty, somewhat wrinkly, brownish grey (4F2),arising aerial hyphae at the center (pale grey (4B1) to brownish grey(4F2)) R: Dark grey (4F1) Czapeck's solution G: Very restrictedly,1.0-1.5 cm agar (Raper and Thom S: Irregular, thin, scanty, 1949)immersed, subhyaline to white R: Subhyaline to white Sabouraud dextroseG: Restrictedly, 2.0-2.5 cm agar (Difco 0109) S: Circular, plane, thin,white, sectoring, light brown (6D5) at the colony center R: Pale yellow(4A3) Oatmeal agar G: Fairly rapidly, 4.0-4.5 cm (Difco 0552) S:Circular, plane, felty to cottony, dark grey (4F1) to brownish grey(4F2) R: Brownish grey (4D2) Emerson Yp Ss agar G: Restrictedly, 2.0-2.5cm (Difco 0739) S: Circular to irregular, plane, felty, dark grey (4F1)to brownish grey (4F2) R: Medium grey (4E1) to dark grey (4F1) Corn mealagar G: Rather restrictedly, 2.5-3.0 cm (Difco 0386) S: Circular, plane,thin to felty, dark grey (2F1) to olive (2F3) R: Dark grey (2F1) toolive (2F3) MY20 agar G: Restrictedly, 1.5-2.0 cm S: Circular toirregular, thin, sectoring, yellowish white (4A2) R: Pale yellow (4A3)to orange white (5A2) Abbreviations: G: growth, measuring colony size indiameter S: colony surface R: reverse

These characteristics were observed after 14 days of incubation at 25°C. The color descriptions were based on the Methuen Handbook ofColour⁵⁾.

1) Miura, K. and M. Y. Kudo: An agar-medium for aquatic Hyphomycetes.,Trans. Ycolo. Soc. Japan, 11:116-118, 1970.

2) Arx, J. A. von: The Genera of Fungi—Sporulating in Pure Culture (3rded.), 315 p., J. Cramer, Vaduz, 1974.

3) Sutton, B. C.: The Coelomycetes—Fungi Imperfecti with Pycnidia,Acervuli and Stromata., 696 p., Commonwealth Mycological Instritute,Kew, 1980.

4) Hawksworth, D. L., B. C. Sutton and G. C. Ainsworth: Dictionary ofthe Fungi (7th ed.), 445 p., Commonwealth Mycological Institute, Kew.,1983.

5) Kornerup, A. and Wanscher, J. H.: Methuen Handbook of Colour (3rded.), 252 p., Methuen, London, 1983.

A culture of Coelomycetes strain F-11899 thus named has been depositedwith National Institute of Bioscience and Human-Technology Agency ofIndustrial Science and Technology (former name: Fermentation ResearchInstitute Agency of Industrial Science and Technology) (1-3, Higashi1-chome, Tsukuba-shi, IBARAKI 305 JAPAN) on Oct. 26, 1989 under thenumber of FERM BP-2635.

After that, however, we have further studied the classification of thestrain F-11899, and have found that the strain F-11899 resembledColeophoma empetri (Rostrup) Petrak 1929 ^(2), 3), 4)) belonging to theorder Coelomycetes, but differed in some pycnidial characteristics:globose or flattened at the base, immersed, and not papillate.

Considering these characteristics, we classified this strain in moredetail and renamed it as “Coleophoma sp. F-11899”.

In this connection, we have already taken step to amend the name,“Coelomycetes strain F-11899” to “Coleophoma sp. F-11899” with NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology (former name: Fermentation Research InstituteAgency of Science and Technology, on Sep. 21, 1990.

(ii) Production of the compound [Ia] or a salt thereof.

The compound [Ia] or a salt thereof of this invention is produced whenthe compound [Ia] or a salt thereof-producing strain belonging to thegenus Coleophoma is grown in a nutrient medium containing sources ofassimilable carbon and nitrogen under aerobic conditions (e.g. shakingculture, submerged culture, etc.) in the presence of leucine. It isdesirable to contain leucine between about 0.05% and 5% (more preferablybetween about 0.1% and 1%) in a nutrient medium.

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, sucrose, starch, fructose or glycerin, or the like.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cotton seed flour, soybean meal, corn steep liquor, dried yeast,wheat germ, etc., as well as inorganic and organic nitrogen compoundssuch as ammonium salts (e.g. ammonium nitrate, ammonium sulfate,ammonium phosphate, etc.), urea or amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not to be used in their pure form because less purematerials, which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such assodium or calcium carbonate, sodium or potassium phosphate, sodium orpotassium chloride, sodium or potassium iodide, magnesium salts, coppersalts, zinc salts, or cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously adefoaming agent, such as liquid paraffin, fatty oil, plant oil, mineraloil or silicone, or the like may be added.

As in the case of the preferred methods used for the production of otherbiologically active substances in massive amounts, submerged aerobiccultural conditions are preferred for the production of the compound[Ia] or a salt thereof in massive amounts.

For the production in small amounts, a shaking or surface culture in aflask or bottle is employed.

Further, when the growth is carried out in large tanks, it is preferableto use the vegetative form of the organism for inoculation in theproduction tanks in order to avoid growth lag in the process ofproduction of the compound [Ia] or a salt thereof. Accordingly, it isdesirable first to produce a vegetative inoculum of the organism byinoculating a relatively small quantity of culture medium with spores ormycelia of the organism and culturing said inoculated medium, and thento transfer the cultured vegetative inoculum to large tanks. The medium,in which the vegetative inoculum is produced, is substantially the sameas or different from the medium utilized for the production of thecompound [Ia] or a salt thereof.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 10°C. and 40° C., preferably 20° C. to 30° C., for a period of about 50hours to 150 hours, which may be varied according to fermentationconditions and scales.

When the fermentation is completed, the culture broth is then subjectedfor recovery of the compound [Ia] or a salt thereof to variousprocedures conventionally used for recovery and purification ofbiological active substances, for instance, solvent extraction with anappropriate solvent or a mixture of some solvents, chromatography orrecrystallization from an appropriate solvent or a mixture of somesolvents, or the like.

According to this invention, in general, the compound [Ia] or a saltthereof is found both in the cultured mycelia and cultured broth.Accordingly, then the compound [Ia] or a salt thereof is removed fromthe whole broth by means of extraction using an appropriate organicsolvent such as acetone or ethyl acetate, or a mixture of thesesolvents, or the like.

The extract is treated by a conventional manner to provide the compound[Ia] or a salt thereof, for example, the extract is concentrated byevaporation or distillation to a smaller amount and the resultingresidue containing active material, i.e. the compound [Ia] or a saltthereof is purified by conventional purification procedures, forexample, chromatography on recrystallization from an appropriate solventor a mixture of some solvents.

When the object compound is isolated as a salt of the compound [Ia], itcan be converted to the free compound [Ia] or another salt of thecompound [Ia] according to a conventional manner.

Process 2

The object polypeptide compound [Ib] or a salt thereof can be preparedby subjecting a compound [Ia] or a salt thereof to elimination reactionof N-acyl group.

This reaction is carried out in accordance with a conventional methodsuch as hydrolysis, reduction, reaction with an enzyme or the like.

The hydrolysis is preferably carried out in the presence of a base or anacid including Lewis acid. Suitable base may include an inorganic baseand an organic base such as an alkali metal [e.g. sodium, potassium,etc.], an alkaline earth metal [e.g. magnesium, calcium, etc.], thehydroxide or carbonate or bicarbonate thereof, trialkylamine [e.g.trimethylamine, triethylamine, etc.], picoline,1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]-octane,1,8-diazabicyclo[5.4.0]undec-7-ene, or the like.

Suitable acid may include an organic acid [e.g. formic acid, aceticacid, propionic acid, trichloroacetic acid, trifluoroacetic acid, etc.]and an inorganic acid [e.g. hydrochloric acid, hydrobromic acid,sulfuric acid, hydrogen chloride, hydrogen bromide, etc.]. Theelimination using Lewis acid such as trihaloacetic acid [e.g.trichloroacetic acid, trifluoroacetic acid, etc.], or the like, ispreferably carried out in the presence of cation trapping agents [e.g.anisole, phenol, etc.].

The reaction is usually carried out in a solvent such as water, analcohol [e.g. methanol, ethanol, etc.], methylene chloride,tetrahydrofuran, a mixture thereof or any other solvent which does notadversely influence the reaction. A liquid base or acid can be also usedas the solvent. The reaction temperature is not critical and thereaction is usually carried out under cooling to warming.

The reduction method applicable for the elimination reaction may includechemical reduction and catalytic reduction.

Suitable reducting agents to be used in chemical reduction are acombination of metal [e.g. tin, zinc, iron, etc.] or metallic compound[e.g. chromium chloride, chromium acetate, etc.] and an organic orinorganic acid [e.g. formic acid, acetic acid, propionic acid,trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid,hydrobromic acid, etc.].

Suitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalysts [e.g. platinum plate, spongy platinum,platinum black, colloidal platinum, platinum oxide, platinum wire,etc.], palladium catalysts [e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.], nickel catalysts[e.g. reduced nickel, nickel oxide, Raney nickel, etc.], cobaltcatalysts [e.g. reduced cobalt, Raney cobalt, etc.], iron catalysts[e.g. reduced iron, Raney iron, etc.], copper catalysts [e.g. reducedcopper, Raney copper, Ullman copper, etc.] and the like.

The reduction is usually carried out in a conventional solvent whichdoes not adversely influence the reaction such as water, methanol,ethanol, propanol, N,N-dimethylformamide, or a mixture thereof.Additionally, in case that the above-mentioned acids to be used inchemical reduction are in liquid, they can also be used as a solvent.Further, a suitable solvent to be used in catalytic reduction may be theabove-mentioned solvent, and other conventional solvent such as diethylether, dioxane, tetrahydrofuran, etc., or a mixture thereof.

The reaction temperature of this reduction is not critical and thereaction is usually carried out under cooling to warming.

The reaction with an enzyme can be carried out by reacting the compound[Ia] or a salt thereof with an enzyme suitable for the eliminationreaction of N-acyl group.

Suitable example of said enzyme may include the one produced by certainmicroorganisms of the Streptomyces or the Actinoplanaceae, for example,Streptomyces sp. No. 6907 (FERM BP-5809), Streptomyces anulatus No. 4811(FERM BP-5808), Streptomyces anulatus No. 8703 (FERM BP-5810),Actinoplanes utahensis IFO-13244, Actinoplanes utahensis ATCC 12301,Actinoplanes missenrienses NRRL 12053, or the like; and the like.

This elimination reaction is usually carried out in a solvent such asphosphate buffer, Tris-HCl buffer or any other solvent which does notadversely influence the reaction.

The reaction temperature is not critical and the reaction can be carriedout at room temperature or under warming.

Process 3

The object polypeptide compound [Ic] or a salt thereof can be preparedby reacting the compound [Ib] or its reactive derivative at the aminogroup or a salt thereof with the compound [II] or its reactivederivative at the carboxy group or a salt thereof.

Suitable reactive derivative at the carboxy group of the compound [II]may include an acid halide, an acid anhydride, an activated amide, anactivated ester, and the like. Suitable examples of the reactivederivatives may be an acid chloride; an acid azide; a mixed acidanhydride with an acid such as substituted phosphoric acid [e.g.,dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid,dibenzylphosphoric acid, halogenated phosphoric acid, etc.],dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuricacid, sulfonic acid [e.g., methanesulfonic acid, etc.], aliphaticcarboxylic acid [e.g., acetic acid, propionic acid, butyric acid,isobutyric acid, pivaric acid, pentanoic acid, isopentanoic acid,2-ethylbutyric acid, trichloroacetic acid, etc.]; or aromatic carboxylicacid [e.g. benzoic acid, etc.]; a symmetrical acid anhydride; anactivated amide with imidazole, 4-substituted imidazole,dimethylpyrazole, triazole, tetrazole or 1-hydroxy-1H-benzotriazole; oran activated ester [e.g., cyanomethyl ester, methoxymethyl ester,dimethyliminomethyl [(CH₃)₂N⁺═CH—] ester, vinyl ester, propargyl ester,p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester,pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester,phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester,carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester,8-quinolyl thioester, etc.], or an ester with a N-hydroxy compound [e.g.N,N-dimethylhydroxylamine, 1-hydroxy-1-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-1H-benzotriazole,etc.], and the like. These reactive derivatives can optionally beselected from them the mind of the compound [II] to be used.

Suitable salts of the compound [II] and its reactive derivative can bereferred to the ones as exemplified for the object polypeptide compound[I].

The reaction is usually carried out in a conventional solvent such aswater, alcohol [e.g., methanol, ethanol, etc.], acetone, dioxane,acetonitrile, chloroform, methylene chloride, ethylene chloride,tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or anyother organic solvent which does not adversely influence the reaction.These conventional solvent may also be used in a mixture with water.

In this reaction, when the compound [II] is used in a free acid form orits salt form, the reaction is preferably carried out in the presence ofa conventional condensing agent such as N,N′-dicyclohexylcarbodiimide;N-cyclohexyl-N′-morpholinoethylcarbodiimide;N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide;N,N′-diethylcarbodiimide, N,N′-diisopropylcarbodiimide;N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide,N,N-carbonylbis-(2-methylimidazole);pentamethyleneketene-N-cyclohexylimine;diphenylketene-N-cyclohexylimine; ethoxyacetylene;1-alkoxy-2-chloroethylene; trialkyl phosphite; ethyl polyphosphate;isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride);phosphorus trichloride; thionyl chloride; oxalyl chloride; lower alkylhaloformate [e.g., ethyl chloroformate, isopropyl chloroformate, etc.];triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt;2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide intramolecular salt;1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; so-calledVilsmeier reagent prepared by the reaction of N,N-dimethylformamide withthienyl chloride, phosgene, trichloromethyl chloroformate, phosphorusoxychloride, methanesulfonyl chloride, etc.; or the like.

The reaction may also be carried out in the presence of an inorganic ororganic base such as an alkali metal carbonate, alkali metalbicarbonate, tri(lower)alkylamine, pyridine, di(lower)alkylaminopyridine(e.g., 4-dimethylaminopyridine, etc.), N-(lower)alkylmorpholine,N,N-di(lower)alkylbenzylamine, or the like.

The reaction temperature is not critical, and the reaction is usuallycarried out under cooling to warming.

The compounds obtained by the above Processes 1 to 3 can be isolated andpurified by a conventional method such as pulverization,recrystallization, column-chromatography, high-performance liquidchromatography (HPLC), reprecipitation, or the like.

The compounds obtained by the above Processes 1 to 3 may be obtained asits hydrate, and its hydrate is included within the scope of thisinvention.

It is to be noted that each of the object polypeptide compound [I] mayinclude one or more stereoisomer such as optical isomer(s) andgeometrical isomer(s) due to asymmetric carbon atom(s) and doublebond(s) and all such isomers and mixture thereof are included within thescope of this invention.

Biological Property of the Polypeptide Compound [I] of the PresentInvention

In order to show the usefulness of the polypeptide compound [I] of thepresent invention, the biological data of the representative compound isexplained in the following.

Test (Antimicrobial activity):

Test Compound The object compound of Example 1

Test Method:

Antimicrobial activity of the object compound of Example 1 wasdetermined by a serial broth dilution method using 96-well microtiterplate in 100 μl of MEM (Eagle's minimum essential medium) for Candidaalbicans and in 100 μl of yeast nitrogen base dextrose medium for bothAsperaillus fumigatus and Cryptococcus neoformans. The inoculum wasadjusted to 1×10⁵ colony forming units/ml. Candida albicans andAsperaillus fumigatus were cultured at 37° C. for 24 hours andCryptococcus neoformans was cultured at 37° C. for 48 hours in 5% CO₂incubator. After incubation, the growth inhibition of microorganisms ineach well was determined by microscopic observation. The results wereshown as MEC (minimum effective concentration: μg/ml) value (Table 2).

Test Result

TABLE 2 Microorganisms MEC (μg/ml) Candida albicans FP633 0.04Aspergillus fumigatus FP1305 0.08

From the test result, it is realized that the object polypeptidecompound [I] of the present invention has an antimicrobial activity(especially, antifungal activity).

The pharmaceutical composition of the present invention can be used inthe form of a pharmaceutical preparation, for example, in solid,semisolid or liquid form, which contains the object polypeptide compound[I] or a pharmaceutically acceptable salt thereof, as an activeingredient in admixture with an organic or inorganic carrier orexcipient which is suitable for rectal; pulmonary (nasal or buccalinhalation); ocular; external (topical); oral administration; parenteral(including subcutaneous, intravenous and intramuscular) administrations;insufflation (including aerosols from metered dose inhalator);nebulizer; or dry powder inhalator.

The active ingredient may be compounded, for example, with the usualnon-toxic, pharmaceutically acceptable carriers in a solid form such asgranules, tablets, dragees, pellets, troches, capsules, orsuppositories; creams; ointments; aerosols; powders for insufflation; ina liquid form such as solutions, emulsions, or suspensions forinjection; ingestion; eye drops; and any other form suitable for use.And, if necessary, there may be included in the above preparationauxiliary substance such as stabilizing, thickening, wetting,emulsifying and coloring agents; perfumes or buffer; or any othercommonly may be used as additives.

The object polypeptide compound [I] or a pharmaceutically acceptablesalt thereof is/are included in the pharmaceutical composition in anamount sufficient to produce the desired antimicrobial effect upon theprocess or condition of diseases.

For applying the composition to human, it is preferable to apply it byintravenous, intramuscular, pulmonary, oral administration, orinsufflation. While the dosage of therapeutically effective amount ofthe object polypeptide compound [I] varies form and also depends uponthe age and condition of each individual patient to be treated, in thecase of intravenous administration, a daily dose of 0.01-20 mg of theobject polypeptide compound [I] per kg weight of human being in the caseof intramuscular administration, a daily dose of 0.1-20 mg of the objectpolypeptide compound [I] per kg weight of human being, in case of oraladministration, a daily dose of 0.5-50 mg of the object polypeptidecompound [I] per kg weight of human being is generally given fortreating or preventing infectious diseases.

Especially in case of the treatment of prevention of Pneumocystiscarinii infection, the followings are to be noted.

For administration by inhalation, the compounds of the present inventionare conveniently delivered in the form of an aerosol spray presentationfrom pressurized as powders which may be formulated and the powdercompositions may be inhaled with the aid of an insufflation powderinhaler device. The preferred delivery system for inhalation is ametered dose inhalation aerosol, which may be formulated as a suspensionor solution of compound in suitable propellants such as fluorocarbons orhydrocarbons.

Because of desirability to directly treat lung and bronchi, aerosoladministration is a preferred method of administration. Insufflation isalso a desirable method, especially where infection may have spread toears and other body cavities.

Alternatively, parenteral administration may be employed using dripintravenous administration.

The following Examples are given for the purpose of illustrating thepresent invention in more detail.

EXAMPLE 1

(1) Fermentation:

An aqueous seed medium (30 ml) containing sucrose 4%, cotton seed flour2%, soybean powder 2%, KH₂PO₄ 1.6% and CaCO₃ 0.2% was poured into a 100ml Erlenmeyer flask and sterilized at 120° C. for 30 minutes. A loopfulof Celeophoma sp. F-11899 was inoculated from a slant culture into theflask. The flask was shaken on a rotary shaker (220 rpm, 5.1 cm-throw)at 25° C. for 7 days and then transferred at the rate of 2% to 160 ml ofthe same sterile seed medium in each of six 500 ml Erlenmeyer flasks.The flasks were shaken on a rotary shaker (220 rpm, 5.1 cm-throw) at 25°C. for 5 days. The resultant seed culture was inoculated to 20 l ofsterile production medium consisting of corn meal 4%, glucose 1%, wheatgerm 0.5%, cotton seed flour 1.25%, gluten meal 0.25%, (NH₄)₂SO₄ 0.6%,KH₂PO₄ 1.6%, Na₂HPO₄.12H₂O 1.6%, Zn(SO₄)₂.7H₂O 0.001%, leucine 0.5%,Adekanol LG-109 (deforming agent, Asahi Denka Co., Ltd.) 0.05% andSilicone KM-70 (deforming agent, Shin-Etsu Chemical Co., Ltd.) 0.05% ina 30 l jar fermentor. Fermentation was carried out at 25° C. for 6 daysunder aeration of 20 l/min and agitation of 250 rpm.

The production of active compound in the fermentation broth wasmonitored by HPLC analysis.

(2) Isolation:

The cultured broth (16.5 l) was extracted with 16.5 l of acetone byintermittent mixing. The acetone extract was filtered with an aid ofdiatomaceous earth and concentrated in vacuo to 18 l. The concentratewas passed through a column (1.5 l) of Diaion HP-20 (Mitsubishi ChemicalCo., Ltd.). The column was washed with water and 30% aqueous methanol,and eluted with methanol and 60% aqueous acetonitrile containing 0.2%NaH₂PO₄.2H₂O. 13 l of water was added to the eluate (8 l) and applied ona column (2 l) of YMC gel (ODS-AM 120-S50, YMC Co., Ltd.). The columnwas developed with 40% aqueous acetonitrile containing 0.3%NaH₂PO₄.2H₂O. To the active fractions (1.38 l) were added 460 ml of 0.5%NaH₂PO₄.2H₂O solution and rechromatographed on YMC gel (2 l), which wasdeveloped with the same solvent. The fractions (900 ml) containingObject Compound (1) were collected and concentrated in vacuo to 690 ml.This solution was subjected to preparative HPLC, YMC-packed column(ODS-AM, SH-343-5AM, S-5, 250×20 mm i.d.) with 40% aqueous acetonitrilecontaining 0.3% NaH₂PO₄.2H₂O as mobile phase and flow rate 9.9 ml/min.The active fractions (310 ml) were concentrated in vacuo to 250 ml andapplied to a column rechromatography on preparative HPLC under the samecondition as above. Each of fractions (261 ml) was combined andconcentrated to an aqueous solution in vacuo. This solution was passedthrough a column (30 ml) of Diaion HP-20. The column was washed withwater and eluted with 80% aqueous methanol. After concentration to anaqueous solution, the eluate was lyophilized to give 53.2 mg of ObjectCompound (1) as a white powder.

(HPLC condition); Column: ODS-AM303 (YMC, 250×4.6 mm i.d.); Eluent: 50%aqueous acetonitrile, 0.25% NaH₂PO₄.2H₂O; Flow rate: 1 ml/min.Detection: UV at 210 nm; Retention time: 8.4 min.

The Object Compound (1) as obtained has the following physico-chemicalproperties.

Appearance: white powder; Nature: acid substance; Melting point:165-170° C. (dec.); Specific rotation: [α]_(D) ²³ −23° (C: 0.5,methanol); Molecular formula: C₅₀H₇₉N₈O₂₁SNa; Elemental Analysis:Calcd.: for C₅₀H₇₉N₈O₂₁SNa.6H₂O C, 46.51, H, 7.10, N, 8.68, S, 2.48 (%);Found: C, 46.25, H, 7.40, N, 8.86, S, 2.14 (%); Molecular weight: ESI-MS(m/z)=1161 (M⁺+1); Solubility: soluble: methanol, water; slightlysoluble: ethyl acetate, acetone; insoluble: chloroform; Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction; negative:Dragendorff reaction, Molish reaction; Thin layer chromatography (TLC):

Stationary phase Developing solvent Rf value silica gel*n-butanol:acetic acid:water 0.33 (4:1:2) *Silica Gel 60 F₂₅₄ plate (madeby E. Merck)

Ultraviolet Absorption Spectrum: λ_(max) ^(methanol) (E_(1 cm) ¹%): 201(564), 221 (246), 274 (16.0)_(nm); λ_(max) ^(methanol+0.01)N-NaOH(E_(1 cm) ^(1%)): 204.5 (1081), 246 (52), 284 (15.5)_(nm); Infraredabsorption spectrum: λ_(max) ^(KBr): 3360, 2920, 2850, 1650, 1640, 1520,1440, 1270, 1050 cm⁻¹; ¹H-NMR (CD₃OD, 500 MHz); δ: 0.89 (3H, t, J=7 Hz),1.16 (3H, d, J=7 Hz), 1.23-1.34 (24H, m), 1.57 (2H, m), 1.92-2.02 (4H,m), 2.20 (2H, m), 2.26 (1H, m), 2.38 (1H, m), 2.47 (1H, dd, J=16 and 9.5Hz), 2.74 (1H, dd, J=16 and 4 Hz), 3.72-3.81 (2H, m), 3.90-4.00 (3H, m),4.25-4.29 (2H, m), 4.33-4.39 (2H, m), 4.46 (1H, dd, J=11 and 6.5 Hz),4.51-4.58 (3H, m), 4.93 (1H, d, J=3 Hz), 5.08 (1H, d, J=4 Hz), 5.25 (1H,d, J=3 Hz), 6.84 (1H, d, J=8 Hz), 7.01 (1H, dd, J=8 and 2 Hz), 7.30 (1H,d, J=2 Hz); ¹³C-NMR (CD₃OD, 125 MHz); δ: 14.4 (q), 19.8 (q), 23.7 (t),27.0 (t), 30.3 (t), 30.4 (t), 30.5 (t), 30.6 (t), 30.7 (t), 30.8 (t×5),33.1 (t), 34.6 (t), 34.9 (t), 36.7 (t), 38.4 (t), 39.6 (t), 47.0 (t),51.4 (d), 55.5 (d), 56.9 (d), 57.1 (t), 58.4 (d), 62.4 (d), 68.2 (d),69.7 (d), 70.7 (d), 70.7 (d), 71.4 (d), 74.1 (d), 74.3 (d), 75.5 (d),76.3 (d), 118.2 (d), 123.2 (d), 125.6 (d), 134.5 (s), 141.1 (s), 150.3(s), 169.3 (s), 172.6 (s), 172.7 (s), 172.8 (s), 173.6 (s), 174.4 (s),175.8 (s), 176.9 (s).

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Object Compound (1) has beenidentified and assigned as follows.

EXAMPLE 2

(1) Fermentation of Actinoplanes utahensis

A stock culture of Actinoplanes utahensis IFO-13244 is prepared andmaintained on agar slant. A loopful of the slant culture was inoculatedinto a seed medium consisted of starch 1%, sucrose 1%, glucose 1%,cotton seed flour 1%, peptone 0.5%, soy bean meal 0.5% and CaCO₃ 0.1%.The inoculated vegetative medium was incubated in a 225 ml wide mouthErlenmeyer flask at 30° C. for about 72 hours on a rotary shaker.

This incubated vegetative medium was used directly to inoculate into aproduction medium (20 l) consisted of sucrose 2%, peanut powder 1%,K₂HPO₄ 0.12%, KH₂PO₄ 0.05% and MgSO₄.7H₂O 0.025%. The inoculatedproduction medium was allowed to ferment in a 30 l jar fermentor at atemperature of 30° C. for about 80 hours. The fermentation medium wasstirred with conventional agitators at 250 rpm and aerated at 20 l perminute. The vegetative mycelium was collected from the fermented brothby filtration and once washed with water. The washed mycelium wasdirectly used to obtain the Object Compound (2).

(2) Reaction Condition

To a solution of the Object Compound (1) (10 g) in water (1000 ml) wasadded 250 ml of 1M Na-phosphate buffer (pH 5.8) and 1000 ml of water anda 250 g wet weight of washed mycelium of Actionoplanes utahensisIFO-13244 which was obtained by above fermentation. The reaction wascarried out at 50° C. with stirring for 3 hours. Increase of the ObjectCompound (2) was monitored by analytical HPLC indicated below.

From a 10 g of the Object Compound (1), a 6.8 g of the Object Compound(2) was formed in the reaction mixture.

(HPLC condition); Column: YMC Pack ODS-AM AM303, S-5 120A (250 mm L.×4.6mm I.D., YMC Co., Ltd.); Eluent: 3% aqueous acetonitrile, 0.5%NaH₂PO₄.2H₂O; Flow rate: 1 ml/min. Detection: UV at 210 nm Retentiontime: 6.1 min.

The Object Compound (2) as obtained has the following physico-chemicalproperties.

Appearance: white powder; Melting point: 150-155° C.; Specific rotation:[α]_(D) ²³ −26° (C: 0.5, H₂O); Molecular formula: C₃₄H₅₀N₈O₂₀S;Molecular weight: ESI-MS (m/z)=923 (M⁺+1); Solubility: soluble: water;slightly soluble: methanol; insoluble: ethyl acetate, n-hexane; Colorreaction: positive: iodine vapor reaction, cerium sulfate reaction,ninhydrin; negative: FeCl₃, Molish reaction; Thin layer chromatography(TLC):

Stationary phase Developing solvent Rf value silica gel* 70% 2-propanolaqueous 0.55 solution *Silica Gel 60 F₂₅₄ plate (made by E. Merck)

Infrared absorption spectrum: λ_(max) ^(KBr): 3400, 2940, 1670, 1630,1540, 1520, 1440, 1280, 1250, 1080, 1050, 950 cm⁻¹; ¹H-NMR (D₂O, 500MHz) δ: 1.23 (3H, d, J=6.5 Hz), 1.97-2.13 (3H, m), 2.29-2.53 (5H, m),3.76-3.80 (2H, m), 3.90 (1H, br d, J=12 Hz), 4.02-4.10 (2H, m),4.16-4.21 (3H, m), 4.37-4.42 (2H, m), 4.45-4.48 (2H, m), 4.55 (1H, m),4.60 (1H, m), 4.71 (1H, m), 4.92 (1H, d, J=6 Hz), 5.01 (1H, d, J=3.5Hz), 5.38 (1H, d, J=3 Hz), 7.03 (1H, d, J=8 Hz), 7.09 (1H, dd, J=2 and 8Hz), 7.27 (1H, d, J=2 Hz); ¹³C-NMR (D₂O, 125 MHz) δ: 55.3 (d), 57.0 (d),58.1 (d), 58.4 (t), 60.2 (d), 63.9 (d), 69.4 (d), 70.2 (d), 71.5 (d),72.8 (d), 73.0 (d), 75.2 (d), 77.1 (d), 77.2 (d), 78.1 (d), 120.5 (d),124.7 (d), 128.3 (d), 134.5 (s), 141.6 (s), 151.1 (s), 171.1 (s), 172.2(s), 174.1 (s), 174.4 (s), 174.5 (s), 176.2 (s), 178.4 (s).

From the analysis of the above physical and chemical properties, and theresult of the further investigation of identification of chemicalstructure, the chemical structure of the Object Compound (2) has beenidentified and assigned as follows.

The Starting Compound in the following Examples 3 to 8 and The ObjectCompounds (3) to (8) in the following Examples 3 to 8 are illustrated bychemical formulae as below.

The Starting Compound

(the same in Examples 3 to 8)

The Object Compounds (3) to (8)

In the following Examples, The Object Compound (X) [e.g. The ObjectCompound (3)] means the object compound of Example (X) [e.g. Example(3)].

Example No. R¹ 3

4

5

6

7

8

EXAMPLE 3

To a solution of Starting Compound (150 mg) and4-[4-(4-hexyloxyphenyl)piperazin-1-yl]benzoic acid benzotriazol-1-ylester (81 mg) in N,N-dimethylformamide (3.5 ml) was addeddiisopropylethylamine (0.032 ml), and stirred for 4 hours at ambienttemperature. The reaction mixture was pulverized with ethyl acetate. Theprecipitate was collected by filtration, and dried under reducedpressure. The powder was dissolved in water, and subjected to columnchromatography on ion exchange resin (DOWEX-50WX4 (Trademark: preparedby Dow Chemical)) eluting with water. The fractions containing theobject compound were combined, and subjected to column chromatography onODS (YMC-gel.ODS-AM.S-50 (Trademark: prepared by Yamamura ChemicalLab.)) eluting with 20% acetonitrile aqueous solution. The fractionscontaining the object compound were combined, and evaporated underreduced pressure to remove acetonitrile. The residue was lyophilized togive Object Compound (3).

IR (KBr): 3359, 1668, 1629, 1537, 1510 cm⁻¹; NMR (DMSO-d₆, δ): 0.88 (3H,t, J=6.7 Hz), 1.05 (3H, d, J=5.7 Hz), 1.29-1.5 (6H, m), 1.5-1.71 (2H,m), 1.71-2.04 (3H, m), 2.04-2.4 (4H, m), 2.59 (1H, m), 3.16 (4H, m),3.4-3.7 (1H, m), 3.72 (2H, m), 3.8-4.0 (6H, m), 4.0-4.29 (6H, m),4.29-4.5 (5H, m), 4.74 (2H, d, J=5.7 Hz), 4.8-5.1 (6H, m), 5.16 (1H, d,J=3.2 Hz), 5.22 (2H, d, J=3.5 Hz), 5.47 (2H, d, J=6.2 Hz), 6.73 (1H, d,J=8.5 Hz), 6.83 (2H, d, J=8.5 Hz), 6.92 (1H, s), 6.98 (2H, d, J=3.6 Hz),7.03 (2H, s), 7.30-7.45 (3H, m), 7.82 (2H, d, J=8.5 Hz), 8.07 (1H, d,J=7.2 Hz), 8.27 (1H, d, J=7.2 Hz), 8.43 (1H, d, J=7.2 Hz), 8.84 (1H, s);MASS (m/z): 1330.73 (M+Na⁺); Elemental Analysis Calcd. forC₅₇H₇₇N₁₀O₂₂SNa.22H₂O: C, 40.14, H, 7.15, N, 8.21; Found: C, 40.00, H,5.73, N, 8.20.

The following Object Compounds (4) to (8) were obtained according to asimilar manner to that of Example 3.

EXAMPLE 4

IR (KBr): 3359, 1668, 1650, 1631 cm⁻¹; NMR (DMSO-d₆, δ): 0.89 (3H, t,J=6.7 Hz), 1.17 (3H, d, J=6.0 Hz), 1.3-1.47 (6H, m), 1.5-2.0 (6H, m),2.0-2.4 (5H, m), 3.5-4.2 (12H, m), 4.3-4.5 (4H, m), 4.5-4.75 (4H, m),4.84 (1H, s), 4.9-5.4 (6H, m), 6.64 (2H, d, J=8.0 Hz), 6.83 (2H, m),6.9-7.2 (6H, m), 7.8-8.15 (6H, m), 8.31 (1H, m), 8.78 (1H, m) MASS(m/z): 1285.40 (M-Na⁺); Elemental Analysis Calcd. forC₄₉H₆₉N₈O0₂₂SNa.11H₂O: C, 45.45, H, 5.89, N, 9.64; Found: C, 45.31, H,5.88, N, 9.54.

EXAMPLE 5

IR (KBr): 3363, 1648, 1619, 1506, 1257 cm⁻¹; NMR (DMSO-d₆, δ): 0.91 (3H,t, J=7.1 Hz), 1.09 (3H, d, J=5.9 Hz), 1.29-1.5 (4H, m), 1.6-2.4 (9H, m),2.58 (1H, m), 3.5-4.6 (16H, m), 4.8-5.3 (11H, m), 5.53 (1H, d, J=5.9Hz), 6.73 (1H, d, J=8.3 Hz), 6.83 (1H, d, J=8.3 Hz), 6.85 (1H, s), 7.05(1H, s), 7.12 (2H, d, J=8.9 Hz), 7.25 (1H, s), 7.3-7.4 (2H, m), 7.55(1H, s), 7.85 (2H, d, J=8.9 Hz), 7.9-8.1 (5H, m), 8.29 (1H, d, J=5 Hz),8.85 (1H, s), 8.87 (1H, d, J=8.7 Hz); MASS (m/z): 1254 (M−Na⁺);Elemental Analysis Calcd. for C₅₅H₆₈N₉O₂₃SNa.8H₂O: C, 46.44, H, 5.95, N,8.86; Found: C, 46.30, H, 5.78, N, 8.76.

EXAMPLE 6

IR (KBr): 3359, 1655, 1631, 1537, 1519 cm⁻¹; NMR (DMSO-d₆, δ): 0.91 (3H,t, J=6.7 Hz), 1.09 (3H, d, J=5.7 Hz), 1.29-1.5 (4H, m), 1.5-2.0 (6H, m),2.0-2.4 (3H, m), 2.59 (1H, m), 3.5-3.9 (4H, m),3.9-4.5 (13H, m), 4.78(2H, d, J=5.7 Hz), 4.8-4.95 (2H, d, J=5.7 Hz), 5.02 (3H, s), 5.1-5.25(3H, m), 5.51 (1H, d, J=6.0 Hz), 6.73 (2H, d, J=8.0 Hz), 6.7-6.8 (2H, d,J=9.0 Hz), 7.02 (3H, d, J=9.0 Hz), 7.3-7.5 (4H, m), 7.65 (6H, m), 8.09(1H, d, J=7.2 Hz), 8.29 (1H, d, J=7.2 Hz), 8.50 (1H, d, J=7.2 Hz), 8.84(1H, s); MASS (m/z): 1213.73 (M−Na⁺); Elemental Analysis Calcd. forC₅₄H₆₉N₈O₂₂SNa.17H₂O: C, 42.02, H, 6.73, N, 7.26; Found: C, 41.87, H,5.91, N, 7.28.

EXAMPLE 7

IR (KBr): 3359, 1668, 1632, 1539 cm⁻¹; NMR (DMSO-d₆, δ): 0.86 (3H, t,J=6.7 Hz), 1.05 (3H, d, J=6.7 Hz), 1.27 (10H, m), 1.72 (2H, t, J=6.7Hz), 1.75-2.3 (7H, m), 2.58 (1H, m), 3.61 (1H, m), 3.72 (2H, s), 3.8-4.5(17H, m), 4.73 (2H, d, J=6.0 Hz), 4.8-5.1 (5H, m), 5.1-5.3 (3H, m), 5.47(1H, d, J=6.0 Hz), 6.73 (1H, d, J=8.3 Hz), 6.81 (1H, s), 7.15 (2H, d,J=8.3 Hz), 7.30 (1H, s), 7.2-7.5 (3H, m), 7.73 (2H, d, J=6.4 Hz), 7.86(1H, d, J=8.7 Hz), 8.05 (1H, d, J=8.7 Hz), 8.27 (1H, d, J=8.7 Hz), 8.54(1H, d, J=8.7 Hz), 8.84 (1H, s); MASS (m/z): 1153.69 (M−Na⁺); ElementalAnalysis Calcd. for C₄₉H₆₉N₈O₂₂SNa.11H₂O: C, 42.79, H, 6.67, N, 8.15;Found: C, 42.97, H, 6.64, N, 8.10.

EXAMPLE 8

IR (KBr): 3359, 1668, 1648, 1632, 1537 cm⁻¹; NMR (DMSO-d₆, δ): 0.91 (3H,t, J=6.7 Hz), 1.09 (3H, d, J=6.7 Hz), 1.75 (4H, m), 1.5-2.4 (9H, m),2.59 (1H, m), 3.60 (1H, m), 3.73 (2H, s), 3.8-4.1 (5H, m), 4.1-4.3 (5H,m), 4.3-4.6 (4H, m), 4.78 (2H, d, J=5.8 Hz), 4.8-5.1 (5H, m), 5.17 (1H,d, J=2.9 Hz), 5.23 (2H, t, J=4.9 Hz), 5.52 (1H, d, J=7.5 Hz), 6.74 (1H,d, J=8.2 Hz), 6.83 (2H, d, J=7.3 Hz), 7.04 (3H, d, J=8.2 Hz), 7.29-7.45(3H, m), 7.67 (2H, d, J=8.8 Hz), 7.72 (1H, s), 7.80 (4H, t, J=8.1 Hz),8.01 (2H, d, J=8.1 Hz), 8.08 (1H, d, J=8.6 Hz), 8.30 (1H, d, J=7.8 Hz),8.78 (1H, d, J=7.8 Hz), 8.85 (1H, s); MASS (m/z): 1262.98 (M−Na⁺);Elemental Analysis Calcd. for C₅₈H₇₁N₈O₂₂SNa.8H₂O: C, 48.67, H, 6.13, N,7.83; Found: C, 48.72, H, 6.07, N, 7.72.

What is claimed is:
 1. A polypeptide compound of the following generalformula [I]:SEQ ID NO: 1:

wherein R¹ is hydrogen or acyl group, or a salt thereof.
 2. A compoundof claim 1, wherein R¹ is hydrogen, lower alkanoyl, higher alkanoyl,lower alkenoyl, higher alkenoyl, lower alkoxycarbonyl, higheralkoxycarbonyl, aryloxycarbonyl, arylglyoxyloyl,ar(lower)alkoxycarbonyl, lower alkylsulfonyl, arylsulfonyl,ar(lower)alkylsulfonyl, aroyl substituted with higer alkoxy, aroylsubstituted with heterocyclic group which has aryl having lower alkoxy,aroyl substituted with heterocyclic group which has aryl having higheralkoxy, ar(lower)alkenoyl substituted with aryl having lower alkoxy,ar(lower)alkenoyl substituted with aryl having higher alkoxy, aroylsubstituted with aryl which has aryl having lower alkoxy or aroylsubstituted with aryl which has aryl having higher alkoxy.
 3. A compoundof claim 2, wherein R¹ is hydrogen, higher alkanoyl, aroyl substitutedwith higher alkoxy, aroyl substituted with heterocyclic group which hasaryl having lower alkoxy, aroyl substituted with heterocyclic groupwhich has aryl having higher alkoxy, ar(lower)alkenoyl substituted witharyl having lower alkoxy or aroyl substituted with aryl which has arylhaving lower alkoxy.
 4. A process for preparing a polypeptide compoundof the following general formula [I] (SEQ ID NO: 1):

wherein R¹ is hydrogen or acyl group, or a salt thereof, which comprisesi) fermenting the strain F-11899 belonging to the genus Coleophoma whichis capable of producing a compound of the formula [Ia] or a saltthereof:

 in a nutrient medium in the presence of leucine and recovering thecompound [Ia] or a salt thereof, to give the compound [Ia] or a saltthereof, or ii) subjecting a compound of [Ia] or a salt thereof, toelimination reaction of N-acyl group, to give a compound of the formula[Ib]:

 or a salt thereof, or iii) reacting a compound of [Ib] or a saltthereof, with a compound [II] of the formula R_(a) ¹—OH  [II]  or itsreactive derivative at the carboxy group or a salt thereof, to give acompound of the formula [Ic]:

 wherein R_(a) ¹ is acyl, group, or a salt thereof.
 5. A pharmaceuticalcomposition which comprises, as an active ingredient, a compound ofclaim 1 or a pharmaceutically acceptable salt thereof in admixture witha pharmaceutically acceptable carrier or excipient.
 6. A process for theprophylactic and/or the therapeutic treatment of infectious diseasescaused by pathogenic microorganisms which comprises administering acompound of claim 1 or a pharmaceutically acceptable salt thereof to ahuman being or an animal.
 7. A polypeptide compound according to claim 1wherein R¹ is hydrogen.